Since work on this project begun in February 2017, we have generated a large panel of novel IFITM3 mutants to aid our study. Furthermore, we have developed systems using MLV-based vectors and adapted a pseudotyping procedure to study how various retroviral glycoproteins affect viral susceptibility to IFITM-mediated antiviral activities. Our data thus far suggest that IFITM3 exhibits great breadth with regard to its ability to inhibit retroviral particle infectivity. These findings suggest that these antiviral proteins may impose serious barriers to multiple mammalian retroviruses of medical importance in different species. Specifically, the presence of IFITM3 in virus-producing cells leads to decreased levels of envelope glycoprotein, which results in virus particles displaying very little glycoprotein needed to attach and perform fusion with cell targets. Importantly, our work thus far demonstrates that IFITM3 can perform this activity when expressed ectopically or endogenously, as RNAi and CRISPR/Cas9-mediated gene knockout of IFITM3 in HeLa cells result in longer half-lives of viral glycoproteins in cells. This indicates that IFITM3 negatively impacts the stability of viral glycoproteins via an unknown mechanism. The first clue toward understanding the basis for glycoprotein inhibition was observed using confocal microscopy, which showed that IFITM3 and MLV glycoprotein colocalize in a perinuclear region that is proximal to the Golgi apparatus. We are currently narrowing down the precise nature of this organelle, which will aid in our description of how protein half-life is inhibited. We are also assessing the specificity of this effect--that is, whether IFITM3 also affects the production, processing, and/or stability of cellular glycoproteins. In parallel, we have identified a number of novel mutations in IFITM3 that disrupt antiretroviral activity. We are currently using structural homology to predict functional motifs to which these mutated sites belong, which will facilitate our understanding of how and why individual mutations disrupt activity. Together, these efforts will provide extensive insight into the function of IFITM proteins and will provide leverage for the development of new anti-HIV therapies.